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J. Biol. Chem., Vol. 282, Issue 1, 337-344, January 5, 2007
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1
From the
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and the Departments of Chemistry and Chemical Biology and of Biology and Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Biotechnology Center, Troy, New York 12180-3590
Received for publication, August 8, 2006 , and in revised form, October 23, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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102-4 sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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1,4-GlcUA-1,3-Glc-NAc-), chondroitin sulfate (-1,4-GlcUA-1,3-GalNAc-), heparan sulfate (
1,4-GlcUA-1,4-GlcNAc-), heparin (-1,4-IdoUA- 1,4-GlcNAc-), and keratan sulfate (-1,3-Gal-1,4-GlcNAc-), are sulfated except for HA. The bacterial GAG polymers are not known to be sulfated. The enzymes involved in the biosynthesis of all the GAG backbones, except for keratan, have been molecularly cloned.
Certain bacterial enzymes, including the Gram-negative Pasteurella multocida type A hyaluronan synthase, PmHAS, and the type F chondroitin synthase, PmCS, are particularly amenable to study because of their two active center architecture (10, 11), their ability to polymerize long chains in vitro (12), and their ability to elongate exogenously supplied acceptor oligosaccharides in vitro (13, 14). The Escherichia coli K4 chondroitin polymerase, KfoC (15), is 60% identical to the PmHAS and PmCS; therefore, this system probably operates in a similar fashion. The streptococcal and animal HA synthase enzymes, however, do not readily utilize exogenously supplied acceptors and are more difficult to study (13). Heparosan synthases from Pasteurella type D, PmHS1 (16), and Pasteurella types A, D, and F, PmHS2 (17), promise to be interesting experimental models as well, but their overall primary structure differs substantially from PmHAS and PmCS.
The PmHAS and PmCS enzymes each possess independent hexosamine and glucuronic acid transfer sites as assessed by mutating various sequence motifs (10, 11). Kinetic studies suggest that there is a separate acceptor binding pocket for each of these glycosyltransferase activities that apparently interacts with three or four saccharide units of the nascent HA chain (18). The sugar nucleotide specificity of native sequence enzymes has been evaluated with naturally occurring UDP-sugar donors; only the authentic monosaccharide molecules are transferred efficiently by the native enzymes (e.g. C-4 epimers will not substitute). All known Pasteurella enzymes add single sugars in a repetitive stepwise fashion to the nonreducing terminus of their cognate acceptor oligosaccharides (13, 17, 19). Here we report that PmHAS and PmCS will elongate a range of acceptor molecules in addition to their cognate sugars; this finding sheds light on the nature of the synthase active sites as well as significantly expanding the potential repertoire of oligosaccharide and polysaccharide targets that may now be synthesized.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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80 and 20 kDa, respectively, by autohydrolysis (121 °C, 20 p.s.i., 20 min) to be more comparable with the smaller molecular weight chondroitin sulfates (15-40 kDa) and heparin (17-19 kDa). In addition, 80-kDa monodisperse HA was prepared by synchronized stoichiometrically controlled chemoenzymatic reactions (12). To avoid the heterogeneity problem intrinsic to natural GAG polysaccharides, a series of defined oligosaccharides of known structure and length (Table 1) was prepared as described below and outlined in Table 2.
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Chondroitin 4-Sulfate Trisaccharide (CSbt3)—Chondroitin 4-sulfate from bovine trachea, CSbt (1.0 g), was treated with chondroitin ABC lyase (EC 4.2.2.4 [EC] , Proteus vulgaris; Sigma; 1 unit) in 50 mM Tris-HCl/sodium acetate buffer at 37 °C to obtain a mixture of unsaturated oligosaccharides. The resulting oligosaccharide products were fractionated on a Bio-Gel P6 (Bio-Rad) column eluted with 100 mM sodium chloride. Sized oligosaccharides were then desalted by SEC on a Bio-Gel P2 column. Charge separation of the tetrasaccharide fraction was carried out by semi-preparative strong anion exchange-high performance liquid chromatography (SAX-HPLC) (21). The major peaks were pooled, lyophilized, and desalted on a Bio-Gel P2 column. The unsaturated chondroitin sulfate tetrasaccharide containing two 4-O-sulfonated GlcNAc residues (1 mg) was dissolved in distilled water (1 mg/ml). To remove the nonreducing terminal unsaturated GlcUA residue, equal volumes of the oligosaccharide solution and mercuric acetate reagent (35 mM, adjusted to pH 5 using acetic acid in distilled water) were added and stirred for 15 min at room temperature. The reaction mixture was then passed through a pre-washed Dowex 50W-X8 H+ column, neutralized with saturated sodium bicarbonate solution, and then applied to a Bio-Gel P2 column to obtain the pure trisaccharide CSb3. The structure of this trisaccharide was confirmed by ESI-MS analysis (Table 3) (21).
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C-5-epimerized Chondroitin 4-Sulfate Trisaccharide and Pentasaccharide (CSpII3 and CSpII5)—C-5-epimerized chondroitin sulfate from porcine intestinal mucosa, CSpII (Celsus; 10 g), was treated with chondroitin ABC lyase (20 units) as above. The resulting oligosaccharide products were fractionated on a Bio-Gel P6 column, and each oligosaccharide was desalted by SEC on a Bio-Gel P2 column. Charge separation of sized oligosaccharide fractions was carried out by SAX-HPLC. The major peaks were pooled, lyophilized, and desalted on a Bio-Gel P2 column. The unsaturated C-5-epimerized chondroitin sulfate tetrasaccharide (6 mg) and hexasaccharide (2 mg) were treated with mercuric acetate reagent to obtain the pure C-5-epimerized chondroitin sulfate trisaccharide, CSpII3, and pentasaccharide, CSpII5. The structures of these oligosaccharides were confirmed by 1H NMR and ESI-MS analysis (Table 3) (21).
Desulfated C-5-epimerized Chondroitin Sulfate (dCSpII) Polysaccharide—The sodium salt of C-5-epimerized chondroitin sulfate (high purity dermatan sulfate from porcine intestinal mucosa; Celsus), CSpII (250 mg), was treated with 250 ml of acidic methanol (1.25 ml of acetyl chloride in 250 ml of methanol) for 3 days at room temperature. The recovered solid product (methyl ester of desulfated epimerized chondroitin sulfate) was treated with 10 ml of 0.1 M sodium hydroxide for 24 h at room temperature to obtain free carboxylate, O-desulfated epimerized chondroitin sulfate dCSpII (22). This structure was confirmed by 1H NMR spectroscopy (23).
Desulfated C-5-epimerized Chondroitin Sulfate Trisaccharide (dCSpII3)—The dCSpII polymer (100 mg) was treated with chondroitin ABC lyase (1 unit) as described earlier. The resulting oligosaccharide products were fractionated on a Bio-Gel P6 column, and each oligosaccharide was desalted on a Bio-Gel P2 column. Charge separation of sized oligosaccharide fractions was carried out by SAX-HPLC. The major peaks were pooled, lyophilized, and desalted on a Bio-Gel P2 column. C-5-epimerized chondroitin tetrasaccharide without sulfate groups at Glc-NAc residues (1 mg) was treated with mercuric acetate reagent to obtain the trisaccharide. The structure of this trisaccharide was confirmed by ESI-MS analysis (Table 3).
Synthetic Heparin Pentasaccharide (Hep5)—The synthetic analog of the anti-thrombin III binding pentasaccharide, fondaparinux sodium (Arixtra®), was obtained from the pharmacy. The contents of 10 syringes (25 mg in 5 ml of isotonic saline total) were collected and dialyzed (1000-Da cutoff) against water for 2 days at 4 °C and lyophilized to obtain the pentasaccharide Hep5 in quantitative yield.
Heparosan Pentasaccharide (He5)—Heparosan polysaccharide was isolated from E. coli K5 and purified according to the published procedure (24). Using heparin lyase III from Flavobacterium heparinum, a controlled partial depolymerization was performed. The oligosaccharide mixture was size-fractionated using a Bio-Gel P6 column. Each fraction was concentrated and desalted on a Bio-Gel P2 SEC column. The unsaturated hexasaccharide was treated with mercuric acetate reagent to obtain heparosan pentasaccharide He5. Analysis by ESI-MS confirmed the structure of this product (data not shown).
Sugar Quantitation—All carbohydrates were assayed by carbazole method (25) for uronic acid using GlcUA as a standard.
Synthase Purification—The recombinant E. coli cells expressing the truncated, soluble dual-action catalysts PmHAS-(1-703) or PmCS-(46-695) were extracted with the permeabilization agent octylthioglucoside (1% w/v) in 1 M ethylene glycol, 50 mM Tris, pH 7.2, containing protease inhibitors (14). The extract was clarified by centrifugation and applied to a pseudoaffinity column (Tosoh Toyopearl AF-Red-650) equilibrated in the same buffer except 50 mM Hepes was substituted for Tris. The protein eluted with a NaCl gradient (0-1.5 M NaCl for 120 min), and the enzyme peak was determined by Coomassie Blue staining of SDS-polyacrylamide gels. The pooled enzyme (95% pure PmHAS or PmCS) was concentrated with an ultrafiltration device (Amicon Ultra 15 ml, 50-kDa cutoff). The protein was quantitated by the Bradford assay with a bovine serum albumin standard (Pierce).
Synthase Assays and Acceptor Utilization Tests—PmHAS or PmCS-catalyzed polymerization was measured with radiolabeled sugar incorporation assays. UDP-GlcUA and UDP-Hex-NAc polymerization was recorded by monitoring incorporation of UDP-[3H]GlcUA as denoted in Equation 1,
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1 µg (13 pmol) of enzyme, PmHAS-(1-703) or PmCS-(46-695), and 0.01 nmol to 3.75 nmol of a given acceptor were employed. A "no acceptor" control was used for all analyses; this background value corresponding to de novo polymer initiation was subtracted from the value obtained from the assay with acceptor. Assays with an HA polymer acceptor were employed in every data set to allow for normalization. Polymerization reactions contained 50 mM Tris, pH 7.2, 1 M ethylene glycol, 2 mM MnCl2, 0.05 mM UDP-[3H]GlcUA (1 µCi; PerkinElmer Life Sciences), and 0.2 mM UDP-HexNAc (UDP-GlcNAc for PmHAS or UDP-Gal-NAc for PmCS) and were incubated at 30 °C for either 3 min (polysaccharide acceptors) or 30 min (oligosaccharide acceptors). Reactions were stopped by placing on ice and adding SDS (2% final w/v). Descending paper chromatography was used to separate the unincorporated radiolabel from the elongated acceptors (10). The assays were performed in duplicate and were linear with respect to enzyme concentration and time; less than 5% UDP-sugar substrate was consumed.
Single sugar additions were also monitored by reverse phase HPLC ESI-MS (26). Briefly, similar reaction conditions were utilized as described above, except only single unlabeled UDP-sugar (2-6 mM final) was employed. The enzyme adds a monosaccharide unit onto the nonreducing end of various acceptors as shown in Equation 2 or 3.
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Chimeric GAG Synthesis—The purified enzymes PmHAS-(1-703) or PmCS-(46-695) (1 µg) were used to add unlabeled HA or chondroitin chains, respectively, to various GAG polysaccharide acceptors (8-225 µg). Reactions (25 µl) contained the same reaction buffer as above except that 2-4 mM UDP-GlcUA and 2-4 mM UDP-HexNAc (UDP-GlcNAc for PmHAS or UDP-GalNAc for PmCS) were utilized at 30 °C for varying times (see Equation 1, but without the 3H label).
Size Analysis of Polysaccharides—Polymers were analyzed using 1-1.2% 1x TAE-agarose gels (30 V, 5 h, Stains-All detection) (27). The specific Streptomyces hyaluronate lyase from Sigma was employed to destroy and thus identify authentic HA chains. Defined HA molecular weight standards were from (Hyalose L.L.C., Oklahoma City, OK) (12). Kilobase DNA standards were from Stratagene (La Jolla, CA).
Analytical high performance SEC was performed with PLaquagel-OH 60, -OH 50, -OH 40 columns in tandem (15 µm, 7.5 x 300 mm, Polymer Laboratories Amherst, MA) eluted with 50 mM NaPO4, 150 mM NaCl, pH 7, at 0.4-0.5 ml/min. Multiangle laser light scattering (MALLS) detection was performed to quantify absolute molecular weights (12).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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The conundrum of an impure bulk GAG polymer population found in natural extracts was addressed here by employing defined (i.e. HPLC-purified, mass spectrometry, and NMR-validated) oligosaccharides with known sulfation patterns. The nature of the sulfation pattern seems to be an important characteristic on the extent of utilization of the modified acceptor by both PmCS and PmHAS. For example, the 6-sulfated chondroitin trisaccharide (CSs3) and chondroitin pentasaccharide (CSs5) served as relatively good acceptors for PmHAS. In contrast, the 4-sulfated chondroitin trisaccharide (CSbt3) preparation was a poor acceptor (Table 5). In fact, the signals in the radiolabeled incorporation assay of CSbt3 appear to be due entirely to a trace amount of pentasaccharide with mixed 4- and 6-sulfates contaminating the preparation based on ESI-MS of the PmHAS-extended mixture. The starting CSbt3 trisaccharide peak (calculated 760.12 Da; observed 759.1) was not extended into the predicted tetrasaccharide product (calculated 936.15 Da); a major fraction of the pentasaccharide, however, was extended into a hexasaccharide possessing the appropriate mass.
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Two other sulfated chondroitin polysaccharides containing GalNAc-6SO4, CSsf and CSq, show some activity as acceptors (Table 4), but both CSsf and CSq also have an additional sulfate group; CSsf contains GlcUA-2SO4, whereas CSq contains Gal-NAc-6/4-disulfate. These polymers serve as modest acceptors (2.4 and 5.1%, respectively, for PmHAS) but are not as good as CSbt (12%) and CSs (10%). Again, the relative activity of the various natural chondroitin preparations to HA may be affected by the fact that these polymers are not 100% pure and can contain additional minor sequences at the nonreducing termini.
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Desulfated C-5-epimerized chondroitin sulfate B (dCSpII) was also assayed to determine whether the IdoUA or sulfate groups (Fig. 1) were the problematic structures for the synthases. The desulfated C-5-epimerized polysaccharide, dCSpII, showed 2.4% activity relative to HA, higher than that of the 4-sulfated epimerized chondroitin polymers (CSpI and CSpII, 0.4 and 0.5%, respectively; see Table 4) but lower than that of the unsulfated C polymer (54%). We hypothesized that PmHAS and PmCS may be able to tolerate IdoUA to some degree due to the increase in signal in dCSpII (after de-sulfation) when compared with CSpI and CSpII. There is a chance, however, that IdoUA was not well tolerated. The observed signal may have actually been because of the nonreducing terminus of some chains in the polysaccharide population containing an accessible GlcUA, the native uronic acid for the synthase, instead of IdoUA (i.e. CSpII, the original material that was de-sulfated to make dSCpII, contains 95% IdoUA, and thus there is an 5% chance of the GlcUA residing at the nonreducing terminus of the polymers). To further address this issue, a defined IdoUA-containing trisaccharide, dCSpII3, was subjected to a single sugar addition reaction (Eq. 2) with PmHAS. Reverse phase HPLC-ESI-MS analysis verified that the trisaccharide (calculated 600.2 Da; observed 599.2 Da) was converted to the expected tetrasaccharide (calculated 776.23 Da; observed 775.1 Da). Therefore, IdoUA is tolerated by PmHAS. An exact comparison of the GlcUA versus IdoUA utilization rates will need to await the preparation of larger amounts of defined dCSpII3 oligosaccharide. The difference between the GlcUA and the IdoUA epimers is the conformation of the sugar ring. Even though both monosaccharide units have the carboxylate in the equatorial position, two different chair forms (4C1 versus 1C4 for GlcUA or IdoUA, respectively) are present and changes the linkage from to upon epimerization.
Two antithrombotics, the synthetic heparin pentasaccharide, Arixtra (Hep5), and bulk heparin polysaccharide (Hep), show very low (0.03%, Hep) relative acceptor activity to HA or were inhibitory (-0.1%, Hep5) with PmHAS (Tables 4 and 5). The unsulfated heparosan oligosaccharide (1.6%, He5) and polysaccharide (0.1%, H) tested show slightly more activity than the sulfated heparin oligosaccharide and polysaccharide, respectively. It may be that the repeated GlcUA groups among all acidic GAGs can partially compensate for the improper sugar backbone linkages, albeit poorly. However, too many additional negative groups (i.e. sulfate groups) may also be detrimental.
Overall, PmCS exhibited similar acceptor specificity relative to PmHAS (Tables 4 and 5) except that the various chondroitin oligosaccharides were better acceptors for the chondroitin synthase, perhaps reflecting its optimization for chondroitin biosynthesis. A few minor potential differences among the various acceptors were noted, but at this time limiting amounts of sugar reagents precluded further ranking.
Perhaps it is unexpected that the PmHAS and PmCS enzymes would elongate noncognate or sulfated GAGs, but it is not illogical. Some glycosyltransferases have been reported to be promiscuous with respect to their substrate recognition. Also, in living bacteria, the synthase enzymes in the cell interior are not expected to be confronted with noncognate acceptor molecules; thus these catalysts do not need to be selective. On the other hand, the synthase donor sites (especially the UDP-hexosamine pocket) are exposed to structurally similar UDP-sugars, and thus more stringent selectivity for the authentic substrate is expected and is observed.
A potential biotechnological utility of the relaxed acceptor usage (i.e. the ability of these particular synthases/polymerases to accept unnatural substrates) is the creation of GAG molecules with two or more types of sugar repeating units. A variety of chimeric polymers consisting of two distinct GAG polysaccharides fused together were produced (Figs. 2 and 3). The synthase-catalyzed reactions were rapid (complete within 2-6 h). The length of the added HA or chondroitin chain may be controlled by altering the stoichiometry of UDP-sugar to acceptor (12). For example, chondroitin sulfate was extended by PmHAS with HA chains ranging from 400 kDa (2,000 saccharides; Fig. 3) to 1,600 kDa (8,000 saccharides) depending on the donor/GAG acceptor ratio. In addition, the chimeric polymers appear to be relatively monodisperse populations (for example, in Fig. 3, polydispersity Mw/Mn values: HA-CSbt, 1.006 ± 0.02; C-HA, 1.003 ± 0.02; for reference, "1" is the ideal polymer preparation).
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We did note that the entire bulk chondroitin sulfate polymer starting material was not incorporated into chimeric molecules even after repeated addition of synthase and UDP-sugars; this observation suggests that certain nonreducing termini (especially the 4-sulfated chondroitin units) cannot be efficiently extended by native sequence PmHAS or PmCS enzymes. At the start of a reaction utilizing a poorly functioning noncognate acceptor, the Pasteurella synthase slowly extends the polymer, but once the catalyst adds on a short cognate sugar extension to the nonreducing terminus, the nascent molecule is transformed rapidly into an excellent acceptor. At longer times (e.g. approximately >2 h), the differences because of the initial lag period are obscured, and thus the differences among the various chondroitin sulfate preparations are less apparent.
The only other reported methods for chemoenzymatically constructing chimeric GAGs involved the use of testicular hyaluronidase to (a) transglycosylate GAG chains (28, 29) or (b) couple oxazoline monomers (30). Testicular hyaluronidase catalyzes the cleavage of HA, chondroitin, or chondroitin sulfate using a water hydroxyl with an optimum at pH 5. However, the hyaluronidase can also use a polymer hydroxyl group thus adding saccharide units instead of cleaving in a reaction called transglycosylation with an optimum at pH 7. Unfortunately, the transglycosylation method is difficult to control and has low yields; a mixture of products (i.e. a series of structurally similar chimeric GAGs with 6-22 saccharide units) result, and the degrading enzyme will actually cleave the products. In the latter elegant method, oxazoline sugar analogs (e.g. employing mixtures of HA, chondroitin, or chondroitin sulfate disaccharides, etc.) mimicking the transition state are coupled by the hyaluronidase. However, these analogs are unstable in water resulting in a potential overabundance of "dead-end" acceptors (the oxazoline analog degrades to an ordinary disaccharide) compared with the activated donor that drives down the size distribution of the final products (10-20 kDa or 50-100 sugars reported). In addition, the product degradation by hyaluronidase problem mentioned above in method a also exists. Also, the exact placement of desired sugars within a given target structure (especially for small chains) is difficult or impossible for the oxazoline process. On the other hand, the HA and chondroitin synthases and their mutants may be utilized in a step-wise fashion to make defined GAG oligosaccharides (14).
Proteoglycan-like molecules with two distinct high molecular weight GAG components, but missing the protein bridge (e.g. core and link proteins), can be assembled by our method. These molecules may potentially serve the field of tissue engineering as scaffolds to assemble various cell types into tissues or organs. The lack of any polypeptide in these artificial chimeric molecules removes the issues of protease susceptibility and concerns of both immunogenicity and allergenicity (e.g. when an animal-derived product is used in humans). Furthermore, the threat of adventitious agents (e.g. prions, virus) from animal sources may be diminished or eliminated.
On a smaller scale, novel oligosaccharides with HA-like and/or chondroitin-like structures may also be constructed using the Pasteurella synthases.3 We are exploring if these molecules bind with different selectivity or affinity to hyaladherins, HA-binding proteins, or receptors. In addition to mapping out the oligosaccharide binding requirements of hyaladherins, we may be able to generate compounds that will selectively inhibit the binding of HA to one particular hyaladherin species without perturbing other species. Such sugar molecules may have future utility as selective therapeutics with minimal side effects for diseases such as cancer, autoimmune disease, inflammation, and infection.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.
1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73104. Tel.: 405-271-2227; Fax: 405-271-3092; E-mail: paul-deangelis{at}ouhsc.edu.
2 The abbreviations used are: GAG, glycosaminoglycan; PmHAS, P. multocida hyaluronan synthase; HA, hyaluronan, hyaluronate, or hyaluronic acid; C, chondroitin; PmCS, P. multocida chondroitin synthase; HexNAc, N-acetylhexosamine; SEC, size exclusion chromatography; SAX-HPLC, strong anion exchange-high performance liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; Me2SO, dimethyl sulfoxide; Hep, heparin; MALLS, multiangle laser light scattering.
3 B. S. Tracy, A. E. Sismey, and P. L. DeAngelis, unpublished observations.
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ACKNOWLEDGMENTS |
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